The goal of this research project is to identify, genome-wide, the genes and genetic networks controlling retinal cell development, which will form the basis for further studies designed to improve our ability to understand, diagnose, treat and prevent human retinal diseases. Using the fruit fly Drosophila melanogaster as an animal model system, genes and pathways regulated by eyeless (ey), a Pax6 homolog that functions near the top of the genetic hierarchy controlling retinal cell fate specification will be identified using a combinatorial approach of genetics, genomics, and computational biology. In addition, this novel combinatorial approach, once established, can be applied to studies of other transcription factors during retinal development. A Pax6 homolog, ey functions as a master control gene that is both essential and sufficient for eye development. However, only one direct target of ey, sine oculis (so), has been identified. As an essential step toward a full understanding of ey function, a genome-wide screen for its direct downstream targets using three independent methods will be conducted. Gene expression profiles as well as computational approaches have been used to identify several hundred novel gene candidates regulated by ey. To complement the first two approaches, chromatin profiling experiments will be conducted to identify Ey binding sites in the Drosophila genome. Further studies of direct downstream effectors of Ey, as well as other novel genes acting at different levels of the genetic hierarchy identified in this study, will provide us with a comprehensive understanding of the mechanisms of retinal development. Our Specific Aims are: 1. Identification of direct downstream targets of Eyeless during Drosophila retinal development 2. Genome-wide identification of Ey binding sites in the Drosophila genome 3. Functional characterization of novel genes during Drosophila eye development Studies of genes identified in this study will provide the basis for a more complete understanding of eyeless function as well as mechanisms of eye development. Since both ey and many of its downstream genes are highly conserved in humans and many developmental parallels exist between Drosophila and mammalian retina, this work will directly impact our understanding of human retinal development.